CN109735438A - A kind of external system and its application method for reappearing enteric microorganism group - Google Patents
A kind of external system and its application method for reappearing enteric microorganism group Download PDFInfo
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Abstract
The invention belongs to bionics, Human physiology, microbiology, nutrition and analysis and testing technology fields, provide the system and its experimental method of a kind of conditions in vitro reproduction gastrointestinol microorganism group.By sampler, simulator, injection device, culture apparatus, in which: sampler is needle tube type sampler；Simulator includes the anaerobism pipe that bottom is provided with ultrafiltration membrane and is placed in centrifuge tube, and anaerobism tube top portion is arranged butyl rubber plug, nitrogen conduit is arranged on butyl rubber plug and air conduit, nitrogen conduit stretch to anaerobism tube top portion；Air conduit stretches to anaerobism bottom of the tube；Injection device is continuous syringe；Culture apparatus is constant-temperature table.Simulation system is miniature, reaches high-throughput technical requirements.Stomach and intestine simulation carries out in the same pipe, is equipped with ultrafiltration membrane, periodically to simulation pipe centrifugation, simulates the absorption of small intestine, can achieve the effect that Imitative gastroenteric environments.
Description
Technical field
The invention belongs to bionics, Human physiology, microbiology, nutrition and analysis and testing technology fields, specifically relate to
And a kind of external system and its application method for reappearing enteric microorganism group reappears gastrointestinal tract using excrement in vitro
The system and device of microorganism group.Be conducive to multiple samples due to the system and device is miniature because belonging to while being cultivated, to reach
High throughput reappears the technical requirements of gastrointestinal microorganisms group.
Background technique
As a part of intact organism, intestinal flora is other than participating in maintaining body health activity, they are also and very
The occurrence and development of more diseases have close correlation.The physiology and pathological state of intestinal flora and host all have very closely
Relationship.Intestinal flora is to the nutrition of host, metabolism, the growth of epithelial cell and differentiation, the adjusting of immune function and protection
Host play the role of from external world's invasion aspect it is very important, while certain diseases of they and host also have it is close
System.
Type, quantity and the distribution of normal intestinal flora be it is metastable, by diet, endocrine, hygienic habit, geographical ring
Border, the influence at age and sanitary condition and change.It is generally believed that the faecal microbiota of healthy human body and each position flora of enteron aisle form
It is almost the same.Extensive tumour cancer can thus be suffered from and carry out fecal sample acquisition, and these crowds are tracked, using point
Sub- ecological approach carries out dynamic analysis to the gastrointestinal bacterial flora composition of patient during medication, is taking medicine to find cancer trouble
The regularity variation of gastrointestinal bacterial flora in journey.Since the acquisition of fecal sample is the sample collection method of non-damage, to enteron aisle
The composition and quantity of flora carry out dynamic monitoring and are likely to become the weight that a kind of judgement tumour cancer of non-damage suffers from stomach Qi reconstruction
It will foundation.
The research of foundation and method for human gastrointestinal tract bionic system, foreign countries' starting is more early, is also compared by attention degree
It is higher, and the research in China relatively lags behind, mechanism only few in number carries out the research of device and method at present, such as practical
New patent grant number CN201634678U discloses a kind of small-sized fermentation device for simulating intestinal environment, which can be used for reality
Test aerobic room, anaerobism, produce gas, the miniature fermentation such as do not produce gas, can accurately collect with metering ferment caused by gas volume.Invention
Patent authorization number CN102533543B discloses a kind of device that can simulate human body intestinal canal environment for enteric microorganism culture, should
Device can provide and maintain relative constant temperature, humidity and can have certain oscillation, achieve the effect that simulate intestinal environment.
Utility model patent grant number CN202658158U discloses a kind of intelligent biology body pipe intestinal digesting system simulation control dress
It sets, which can be controlled by computer, simulated gastrointestinal tract digestive system.Invention patent mandate CN101482460B is disclosed
A kind of unit animal Bionic digestion system and the method based on the system mock up flat animal digestion, the system and method realize
The process of conditions in vitro Imitating animal gastrointestinal tract digestion and absorption feed.Invention patent mandate CN101665758B discloses one
The simulation experiment method of human gastrointestinal tract and microecosystem is reappeared under kind conditions in vitro, this method is based on big by Ghent, Belgium
The human body intestinal canal microcosm simulation device introduced is learned, the life habit progress technological improvement for east crowd reaches in body
Outer condition reappears human gastrointestinal tract system.Invention patent mandate CN103740589A discloses a kind of bionical system of human gastrointestinal tract
System and the analogue experiment method based on the system, realization under in vitro conditions, can true simulations using the system and method
Human gastrointestinal tract is to the digestion of dietary intake, absorption and intestinal microecology system.
Currently, nobody can guarantee the anaerobic environment of sampling in the patent of domestic applied gastrointestinal tract bionic system, nobody
It can accomplish culture that is miniature, while carrying out multiple samples, and reach high-throughput technical requirements.
Summary of the invention
The object of the present invention is to provide a kind of external systems and its application method for reappearing gastrointestinol microorganism group.
The present invention is realized by following technical solution: the system that a kind of conditions in vitro reappears gastrointestinol microorganism group, by taking
Sampling device, simulator, injection device, culture apparatus, in which: sampler is needle tube type sampler；Simulator includes bottom
The anaerobism pipe that portion is provided with ultrafiltration membrane and is placed in centrifuge tube, anaerobism tube top portion are arranged butyl rubber plug, nitrogen are arranged on butyl rubber plug
Airway and air conduit, nitrogen conduit protrude into bottom in butyl rubber plug, and air conduit stretches to anaerobism bottom of the tube；Injection dress
It is set to continuous syringe；Culture apparatus is constant-temperature table；
The needle tube type sampler is 1ml glass sampler；Anaerobism pipe is 2ml；It is interference fitted at the top of centrifuge tube with anaerobism pipe；Institute
State ultrafiltration membrane be miillpore filter, aperture 0.001um, acid-alkali-resistant degree pH 1.0-2.5,37 DEG C of temperature.
It carries out reappearing gastrointestinol microorganism group using the system that a kind of conditions in vitro reappears gastrointestinol microorganism group
Method, steps are as follows:
(1) it acquires sample: being injected the mixed liquor that gastric juice and food nutrition liquid volume ratio are 2:1 in excrement with glass sample containers,
Mixture is extracted, extracts repeatedly 3 times, finally extracts excrement, gastric juice and food nutrition liquid mixture 0.5ml；
(2) microorganism reappears in stomach: being mixed 1ml food nutrition liquid and gastric juice for the ratio of 2:1 with volume ratio with continuous syringe
In the mixture injection anaerobism pipe of conjunction, adds 0.05M hydrochloric acid and 0.5M sodium bicarbonate, pH are maintained at 1.0-2.5；Butyl rubber plug is close
Anaerobism pipe is sealed, nitrogen 3-5s is then led to, air is discharged, reaches oxygen-free environment in pipe；With sampler by step (1) extracted excrement
Just in mixture injection anaerobism pipe.Playback system is placed on constant-temperature table, setting shaking table temperature is 37 DEG C, frequency of oscillation
200r/min；Incubation time is 2-4h, and every 30min extracts bacterium solution and measures its absorbance value at 600nm in the process, according to survey
Magnitude is at the end of cultivating when OD value is 0.8-1；
(3) culture is completed, and takes bacterium solution sample, and the analysis of microorganism group in stomach is carried out using high-throughput sequencing approach；
(4) microorganism reappears in small intestine: intestinal fluid 0.5ml is added in the anaerobism pipe completed with continuous syringe to culture, is added
0.5M sodium hydroxide, adjusting pH are 6.0-7.5, and it is 37 DEG C that anaerobism pipe, which is then placed in constant-temperature table control shaking table temperature, oscillation
Frequency 100r/min carries out the culture 4-6h of enteric microorganism group；In incubation, starts to cultivate 30min, carry out first time row
Gas in following incubation, is once vented at interval of 1.5h, until culture terminates.Every time after the completion of exhaust, by device
It is placed on centrifuge and carries out centrifugally operated, centrifugal speed 8000-12000r/min, until not having liquid in pipe.After the completion of centrifugation
Will be to the supplement for carrying out intestinal fluid in pipe, and it is 6.0-7.5 that addition sodium hydroxide, which adjusts pH, after cultivating 4h, every 30min is extracted
Bacterium solution measures its absorbance value at 600nm, is at the end of cultivating according to measured value, when OD value is 0.8-1.
(5) culture is completed, and extracts bacterium solution sample, and the analysis of microorganism group is carried out using high-throughput sequencing technologies；
Wherein: gastric juice the preparation method comprises the following steps: 2g NaCl, 3.2g pepsin, are placed in 100mL volumetric flask, it is dense to be added 36.5%
Then HCl 7mL adds sterile distilled water and is settled to 100mL, prepared gastric juice is placed in 4 DEG C of refrigerators and saves；
Pancreatic juice the preparation method comprises the following steps: 0.9g trypsase, 6g bile salt, 12.5gNaHCO3, are dissolved with sterile distilled water and are used in combination
1000mL volumetric flask constant volume, prepared pancreatic juice are placed in 4 DEG C of refrigerators and save；
Food nutrition liquid the preparation method comprises the following steps: proteose peptone 15g, pancreas casein peptone 10g, soy peptone 3g, yeast extract
5g, powdered beef 2g, digestion serum powder 13.5g, beef liver leaching powder 1.2g, glucose 3g, dipotassium hydrogen phosphate 2.5g, sodium chloride 3g, can
Soluble starch 0.3g, L-cysteine 0.3g, sodium thioglycollate 0.15g are added in 1L distilled water, and pH is adjusted to 7.2,25 DEG C and is gone out
30 min of bacterium.
The present invention mainly reappears the micro-ecological environment of stomach and small intestine.By adjusting cholate ratio, and according to microorganism
Condition of culture adjusts system parameter, can effective simulation large intestine environment.
Compared with prior art, the invention has the following advantages that (1) utilizes needle tube type sampler samples, gastric juice will be housed
Excrement is injected with the mixed liquor of food nutrition liquid, extracts the anaerobic environment for guaranteeing sample collection three times repeatedly.(2) reproducer
Simulation for the anaerobism pipe of 2mL, stomach and small intestine carries out in the same anaerobism pipe.Microbial cultivation device is constant-temperature table, energy
Enough guarantee relative constant temperature, humidity, and adjustable frequency of oscillation, simulate gastrointestinal peristalsis, reaches simulation human intestines and stomach
The effect of road environment.Playback system is miniature, can carry out the culture of multiple samples simultaneously, can reach high-throughput technical requirements.(3)
Playback system is equipped with ultrafiltration membrane, is centrifuged using centrifuge in incubation, it can be achieved that the small molecule metabolite generated to metabolism
Real-time absorption, and avoid the disturbance to intestinal colonisation flora, simulate true to nature, the true suction closer to enteron aisle to metabolite
It receives, the training mode of better than traditional gut simulation device.
Detailed description of the invention
Fig. 1 is sampler schematic diagram；Fig. 2 is to reappear system and device schematic diagram；In figure: 1- sampler；1.1- excrement sample；
1.2- gastric juice, food nutrition liquid and excrement sample suspension；Gastric juice, the food nutrition liquid suspension of 1.3- degassing process；2- anaerobism pipe；
3- ultrafiltration membrane；4- centrifuge tube；5- butyl rubber plug；6- nitrogen conduit；7- air conduit.
Specific embodiment
The system that a kind of conditions in vitro reappears gastrointestinol microorganism group, as shown in Fig. 2, by sampler, simulator,
Injection device, culture apparatus, in which: sampler is needle tube type sampler；Simulator include bottom be provided with ultrafiltration membrane and
The anaerobism pipe being placed in centrifuge tube, anaerobism tube top portion are arranged butyl rubber plug, nitrogen conduit and air conduit are arranged on butyl rubber plug,
Nitrogen conduit protrudes into bottom in butyl rubber plug, and air conduit stretches to anaerobism bottom of the tube；Injection device is continuous syringe；Training
Supporting device is constant-temperature table, using injector head insertion butyl rubber plug to being evacuated in pipe in incubation.
The needle tube type sampler is 1ml glass sampler；Anaerobism pipe is 2ml；Match at the top of centrifuge tube with anaerobism pipe interference
It closes；The ultrafiltration membrane be miillpore filter, aperture 0.001um, acid-alkali-resistant degree pH 1.0-2.5,37 DEG C of temperature.
It carries out reappearing gastrointestinol microorganism group using the system that a kind of conditions in vitro reappears gastrointestinol microorganism group
Method, steps are as follows:
(1) sample acquires: the mixed liquor of gastric juice and food nutrition liquid is injected in excrement with glass sample containers, extracts mixture,
It extracts repeatedly 3 times, finally extracts excrement, gastric juice and food nutrition liquid mixture 0.5ml；The syringe of method of sampling 2mL, dress
Enter the mixed liquor 1mL that gastric juice and food nutrition liquid volume ratio by degassing process are 2:1, is injected into inside excrement, utilizes
Syringe needle carries out small-scale agitation, then by its suspension inhalation syringe, is repeated 3 times.It is final extract excrement sample, gastric juice and
Suspension 1mL of food nutrition liquid or so.
(2) microorganism reappears in stomach: 1ml food nutrition liquid stomach function regulating liquid mixture being injected 2mL anaerobism with continuous syringe
In pipe, adds 0.05M hydrochloric acid and 0.5M sodium bicarbonate, pH are maintained at 1.0-2.5；Butyl rubber plug seals anaerobism pipe, then leads to nitrogen
Gas 3-5s is discharged air, reaches oxygen-free environment in pipe；The extracted excrement mixture of step (1) is injected into anaerobism with sampler
In pipe, playback system is established；Playback system is placed on constant-temperature table, setting shaking table temperature is 37 DEG C, frequency of oscillation 200r/
min；Incubation time is 2-4h, and every 30min extracts a bacterium solution in the process, its absorbance value at 600nm is measured, according to survey
Magnitude is at the end of cultivating when OD value is 0.8-1.
(3) culture is completed, and takes bacterium solution sample, and point that microorganism group is reappeared in stomach is carried out using high-throughput sequencing technologies
Analysis；
(4) microorganism reappears in small intestine: intestinal fluid 0.5ml is added in the anaerobism pipe completed with continuous syringe to culture, is added
0.5M sodium hydroxide, adjusting pH are 6.0-7.5, and it is 37 DEG C that anaerobism pipe, which is then placed in constant-temperature table control shaking table temperature, oscillation
Frequency 100r/min carries out the culture 4-6h of enteric microorganism group；In incubation, starts to cultivate 30min, carry out first time row
Gas in following incubation, is once vented at interval of 1.5h, until culture terminates.Every time after the completion of exhaust, by device
It is placed on centrifuge and carries out centrifugally operated, centrifugal speed 8000-12000r/min, until not having liquid in pipe.After the completion of centrifugation
Will be to the supplement for carrying out intestinal fluid in pipe, and adjusting pH by adding sodium hydroxide is 6.0-7.5, after cultivating 4h, every 30min is taken out
A bacterium solution is taken, its absorbance value at 600nm is measured, is at the end of cultivating according to measured value, when OD value is 0.8-1.
(5) culture is completed, and extracts bacterium solution sample, and the analysis of microorganism group is carried out using high-throughput sequencing technologies.
Wherein: gastric juice the preparation method comprises the following steps: 2g NaCl, 3.2g pepsin, are placed in 100mL volumetric flask, be added
Then 36.5% dense HCl 7mL adds sterile distilled water and is settled to 100mL, prepared gastric juice is placed in 4 DEG C of refrigerators and saves；
Pancreatic juice the preparation method comprises the following steps: 0.9g trypsase, 6g bile salt, 12.5gNaHCO3, are dissolved with sterile distilled water and are used in combination
1000mL volumetric flask constant volume, prepared pancreatic juice are placed in 4 DEG C of refrigerators and save；
Food nutrition liquid the preparation method comprises the following steps: the Nanjing proteose peptone 15g(Mao Jie microorganism Science and Technology Ltd.), pancreas casein
Peptone 10g, soy peptone 3g, yeast extract 5g, powdered beef 2g, digestion serum powder 13.5g, beef liver leaching powder 1.2g, glucose 3g,
Dipotassium hydrogen phosphate 2.5g, sodium chloride 3g, soluble starch 0.3g, L-cysteine 0.3g, sodium thioglycollate 0.15g are added 1L and steam
In distilled water, pH is adjusted to 7.2,25 DEG C of 30 min of sterilizing.
Experimental example 1: the feasibility assessment of system stability
1. research object and method
SPF grades of healthy 10 week old male rat of kind 3, Kunming are selected, 300-400 grams of weight, are tested by Shanxi university of TCM dynamic
Object center provides, experimental animal production licence number: SCXK (Shanxi) 2018-0002, Quality of Experimental Animals quality certification number:
0081670.Entire test is divided into two groups, and first group is acquisition healthy rat excrement, and the stomach and intestine using system progress rat are micro-
The reproduction of biology group, second group directly takes the rat gastrointestinal contents for having acquired excrement sample.Two groups of sample DNAs are extracted, after extraction
DNA purified, then carry out agargel electrophoresis detection.PCR amplification is carried out to the qualified DNA of detection, and amplification is produced
Object carries out quality testing and purifying, is surveyed using one section region of the high-flux sequence platform to two groups of sample flora 16SrDNA
Sequence.Compare the similitude otherness between two groups of gastrointestinal bacterial floras according to sequencing result aggregate analysis, assesses the steady of playback system
Qualitative and feasibility.
Experiment key instrument is shown in Table 1, and experiment main agents are shown in Table 2.
Table 1: experiment key instrument
Table 2: experiment main agents
2. experimental procedure
(1) sample collection:
Pressing rat abdomen stimulates its defecation, clamps rat excrement with the tweezers of sterilizing, is quickly put into the training equipped with rat gastric juice
It supports in base, in case subsequent use.Every rat samples about 1g.
Cervical dislocation puts to death the rat for having acquired excrement sample, pipettes stomach and intestines under gnotobasis, is clamped, be placed on tweezers
In the conical flask of ice, small intestine site then is isolated with sterile scissors.Take out stomach and small intestine contents, and with blade scrape stomach and
Small intestinal mucosa.Liquid nitrogen frozen sample is then stored in -80 DEG C of refrigerators, in case subsequent DNA is extracted.
(2) microorganism group culture
Simulator is sealed with butyl rubber plug, leads to nitrogen by air inlet, inner air tube is discharged from exhaust outlet, makes to reach anaerobic in pipe
Environment.The excrement sample of three rats of acquisition is injected separately into 3 simulators by syringe.The temperature that constant-temperature table is arranged is
37 DEG C, frequency of oscillation 200r/min is set.3 simulators are put into constant-temperature table and are cultivated, after culture starts 2h, every
30min extracts bacterium solution and measures its absorbance at 600nm.When cultivating 3h, OD value is shown near 1, i.e., microorganism is in stomach mould
It cultivates and completes in near-ring border.Intestinal fluid 0.5mL is added in the simulation pipe completed by continuous syringe to culture, adds hydroxide
Sodium (0.5M), adjusting pH value is 7.0.The temperature that constant-temperature table is arranged is 37 DEG C, and setting frequency of oscillation is 100r/min, will be simulated
Device is put into the culture 4-6h that enteric microorganism group is carried out in constant-temperature table.In incubation, starts to cultivate 30min, carry out first
Secondary exhaust in following incubation, is once vented at interval of 1.5h, until culture terminates.It, will every time after the completion of exhaust
Device, which is placed on centrifuge, carries out centrifugally operated, centrifugal speed 8000-12000r/min, until not having liquid in pipe.It has been centrifuged
Will be to the supplement for carrying out food nutrition liquid and intestinal fluid in pipe after, and maintenance pH is 6.0-7.5, after cultivating 4h, every 30min
A bacterium solution is extracted, every 30min extracts a bacterium solution, measures its absorbance value at 600nm, according to measured value, OD value is
It is at the end of cultivating when 0.8-1.
(3) sample microorganism group is tested
Sample genomic dna is extracted using E.Z.N.ATM faeces DNA extracts kit.It, will for the accuracy for guaranteeing result
Gained sample flora DNA carries out purity detecting, and sample undesirable for sample flora DNA carries out DNA again and extracts, right
DNA purifying is carried out in repeatedly extracting underproof sample.Gel sugar detected through gel electrophoresis, using PCR instrument to sample flora
The area 16SDNA V3-V6 carries out PCR amplification, tries according to kit specification step QIAquick Gel Extraction Kit
Agent box purifies pcr amplification product.It is sequenced using Roche454 high throughput sequencing technologies.
(4) bioinformatic analysis
Software used in this experimental analysis is QIIME (Quantitative Insights Into Microbial Ecology),
The microbial population analysis software package contains RDP classifier, Py NAST, and Fast Tree etc..OTU
(Operational Taxonomic Units) analysis gathers all sequences that can be used for analyzing using Uclust program
The sequence that similarity is 97% is polymerized to an OUT by class.The analysis of sample α richness is tested by drawing dilution curve,
Dilution curve is to reflect the abundant degree of species in sample indirectly for evaluating whether sequencing amount is enough to cover all monoids.
3. result and analysis
(1) diversity analysis: when synecology microorganisms diversity, usually calculate chao1 index, Simpson index,
Shannon index analyzes the diversity of single sample, and the abundant degree of group, rich in OTU number, chao1 indicial response sample
Richness index is higher, illustrates that the diversity of sample is higher.The diversity of Simpson and Shannon index key reaction group,
It can be influenced by species evenness in sample group and richness.Simpson index is smaller, indicates that diversity is higher.Shannon
Index is bigger, shows that bio-diversity is higher.
Bacterial diversity is analyzed in stomach: the results are shown in Table 3, it is total that the rat stomach microorganism group sample of reproduction obtains high quality sequence
Number 96003, average 32001, each sample, rat stomach content samples obtain high quality sequence sum 112005, average
37335, each sample.It is 193.75 ± 26.02 by system reproduction group OTU number, directly takes the stomach content group OTU number to be
Without significant difference between 192.63 ± 36.12, two groups；It is 203.27 ± 23.03 by system reproduction group chao1 index, directly
Taking stomach content group chao1 index is 208.00 ± 35.45, without significant difference between two groups；Simpson index is reappeared by system
Group is 0.25 ± 0.07, is directly taking gastric content group to be 0.22 ± 0.05, without significant difference between two groups；By system reproduction group
Shannon index is 2.44 ± 0.38, and directly taking stomach content group Shannon index is 2.57 ± 0.29, without significance difference between two groups
It is different.By between richness between two groups and diversity parameters (OTU number, chao1 index, Simpson index, Shannon index)
Comparative descriptions rat excrement sample by playback system group and between take rat gastric content group flora abundance and diversity it
Between without significant difference.
Table 3: the diversity of stomach microorganism is reappeared
Intestinal flora diversity analysis: 4 be the results are shown in Table, it is total that the rat small intestine microorganism group sample of reproduction obtains high quality sequence
Number 112236, average 37412, each sample, rat small intestine content samples obtain high quality sequence sum 132035,
Average 44012, each sample.It is 226.75 ± 26.55 by system reproduction group OTU number, directly takes small intestine content group OTU
Number between 256.63 ± 45.36, two groups without significant difference；It is 236.36 ± 35.03 by system reproduction group chao1 index,
Directly taking small intestine content group chao1 index is 266.37 ± 37.56, without significant difference between two groups；Simpson index is by being
Reproduction group of uniting is 0.35 ± 0.06, is directly taking small intestine contents group to be 0.35 ± 0.04, without significant difference between two groups；By being
Unite reproduction group Shannon index be 2.74 ± 0.58, directly take small intestine content group Shannon index be 2.97 ± 0.29, two groups
Between without significant difference.By diversity parameters between two groups (OTU number, chao1 index, Simpson index, Shannon index) it
Between comparative descriptions rat excrement sample by playback system group and between take rat small intestine content group in the abundance and multiplicity of flora
Without significant difference between property.
Table 4: the diversity of small intestine microorganism is reappeared
(2) sample Bacterial community is analyzed
Bacterial community is analyzed in stomach: analysis of the flora in door categorization levels between two groups the results are shown in Table 5 in stomach, in door level,
We have found that flora is mainly under the jurisdiction of 4 big bacterium doors in two groups of stomaches, including Bacteroidetes (Bacteroidetes), Firmicutes
(Firmicutes), Proteobacteria (Proteobacteria) and actinomyces door (Actinobacteria).It is reappearing in stomach
In flora, Bacteroidetes (60.2%) and Firmicutes (34.9%) have comparative advantage, and are secondly Proteobacteria (3.4%), actinomyces
Door (0.3%)；In gastric content flora, Bacteroidetes (65.2%) and the same proportion of Firmicutes (27.8%) are most, secondly
For Proteobacteria (6.9%), actinomyces door (0.2%).
Table 5: analysis of the flora in door categorization levels between two groups in stomach
Flora reproduction group and gastric content flora group category horizontal analysis the results are shown in Table 6 in stomach, on belonging to level, two groups of floras
For Bacteroides (Bacteroides), flora reproduction group is 48.9% in stomach, and intestinal contents flora group is 53.6%, secondly stomach
The flora of interior flora reproduction group and gastric content group be respectively general Salmonella group (Provetella) (5.1%vs4.8%),Butyricimonas(0.1%vs0.3%),Odoribacter(0.2% vs0.5%), fusobacterium (Clostridium) (0.1%
Vs0.1%),Anaerostipes(0.3% vs0.2%),Blautia (0.5%vs0.4%),Faecalibacterium(4.3%
Vs4.6%), Coprecoccus (Coprococcus) (0.6%vs0.5%),Dorea(0.4% vs0.2%),Roseburia(0.1%
Vs1.6%), Oscillospira (Oscillospira) (0.7%vs0.9%), Lachnospira (Lachnospira) (3.3%
Vs1.6%), Ruminococcus (Rumincoccus) (0vs0.7%), veillonellasp category (Veillonella) (0vs1.5%),
Streptococcus (Streptococcus) (0.7%vs1.0%), Bifidobacterium (Bifidobacterium) (0.7% vs0.8%), Sa
Special Bordetella (Sutterella) (0.9% vs1.5%), thermophilic gallbladder Pseudomonas (Bilophila) (0% vs0.1%).
Table 6: flora reproduction group and gastric content flora group belong to horizontal flora mean value (Mean ± SD) and compare in stomach
Intestinal flora structural analysis: intestinal flora the results are shown in Table 7 in door categorization levels, in door level, it has been found that two groups
Intestinal flora is mainly under the jurisdiction of 4 big bacterium doors, including Bacteroidetes (Bacteroidetes), Firmicutes (Firmicutes), become
Shape bacterium door (Proteobacteria) and actinomyces door (Actinobacteria).In reappearing intestinal flora, Bacteroidetes
(58.2%) it has comparative advantage with Firmicutes (36.9%), is secondly Proteobacteria (2.4%), actinomyces door (0.2%)；In enteron aisle
In tolerant flora, secondly it is Proteobacteria that Bacteroidetes (63.2%) and the same proportion of Firmicutes (26.9%) are most
(8.8%), actinomyces door (0.1%).
Analysis of 7 intestinal flora of table in door categorization levels between two groups
Intestinal flora reproduction group and intestinal contents flora group category level the results are shown in Table 8, and on belonging to level, two groups of floras are
Bacteroides (Bacteroides), intestinal flora reproduction group is 46.9%, and intestinal contents flora group is 51.6%, between two groups
Slightly difference (P=0.07), secondly the flora of intestinal flora reproduction group and intestinal contents group is general Salmonella group respectively
(Provetella) (6.1%vs4.8%),Parabacteroides (2.3%vs2.5%),Butyricimonas(0.3%
Vs0.4%), fusobacterium (Clostridium) (0.1%vs0.1%),Blautia (0.5%vs0.4%),Faecalibacterium (4.3%vs4.6%), Coprecoccus (Coprococcus) (0.6%vs0.5%), Oscillospira (Oscillospira) (0.7%
Vs0.9%), Lachnospira (Lachnospira) (3.3%vs1.6%), Ruminococcus (Rumincoccus) (0.7%
Vs0.7%), koala Bacillus (Phascolarctobacterium) (1.6%vs1.2%), veillonellasp category
(Veillonella) (0%vs1.5%), streptococcus (Streptococcus) (0.7%vs1.0%), Bifidobacterium
(Bifidobacterium) (0.7% vs0.8%), Saudi Bordetella (Sutterella) (0.9% vs1.5%), thermophilic gallbladder Pseudomonas
(Bilophila) (0% vs0.1%).
Table 8: intestinal flora reproduction group and intestinal contents flora group belong to horizontal flora mean value (Mean ± SD) and compare
4. conclusion: the comparison by reappearing rat stomach and rat stomach content bacterial diversity and structure, and reappear rat small intestine
Compared with rat small intestine content bacterial diversity and structure, without the significance difference opposite sex, overall merit should for bacterial diversity and abundance
System feasibility with higher.
Claims (4)
1. the system that a kind of conditions in vitro reappears gastrointestinol microorganism group, it is characterised in that: by sampler, simulator, note
Injection device, culture apparatus, in which: sampler is needle tube type sampler；Simulator includes that bottom is provided with ultrafiltration membrane and sets
In the anaerobism pipe in centrifuge tube, butyl rubber plug is arranged in anaerobism tube top portion, and nitrogen conduit and air conduit, nitrogen is arranged on butyl rubber plug
Airway protrudes into bottom in butyl rubber plug, and air conduit stretches to anaerobism bottom of the tube；Injection device is continuous syringe；Culture
Device is constant-temperature table.
2. the system that a kind of conditions in vitro according to claim 1 reappears gastrointestinol microorganism group, it is characterised in that: described
Needle tube type sampler is 2ml glass sampler；Anaerobism pipe is 2ml；It is interference fitted at the top of centrifuge tube with anaerobism pipe；The ultrafiltration membrane
For miillpore filter, aperture 0.001um, acid-alkali-resistant degree pH 1.0-2.5,37 DEG C of temperature.
3. it is micro- to carry out reproduction gastrointestinal tract using the system that a kind of conditions in vitro described in claim 1 reappears gastrointestinol microorganism group
The method of biology group, it is characterised in that: steps are as follows:
(1) sample acquires: being injected gastric juice and food nutrition liquid according to the mixed liquor 1ml that volume ratio is 2:1 with glass sample containers
It in excrement, is then stirred with syringe needle, extracts suspension, then reinject in excrement, stirred, extract repeatedly, injection 3 times,
Finally extract excrement, gastric juice and food nutrition liquid mixture 0.5ml；
(2) it establishes playback system: the mixture that 1ml food nutrition liquid stomach function regulating liquor ratio is 2:1 being injected into anaerobism with continuous syringe
In pipe, adds 0.05M hydrochloric acid and 0.5M sodium bicarbonate, pH are maintained at 1.0-2.5；Butyl rubber plug seals anaerobism pipe, then passes to
Nitrogen 3-5s is discharged air, reaches oxygen-free environment in pipe；The extracted excrement mixture injection of step (1) is detested with sampler
In oxygen pipe, playback system is established；
(3) microorganism reappears in stomach: playback system being placed on constant-temperature table, control shaking table temperature is 37 DEG C, frequency of oscillation
200r/min；Incubation time is 2-4h, and every 30min extracts bacterium solution and measures its absorbance value at 600nm, root in incubation
Terminate according to measured value OD value to be cultivated when 0.8-1；
(4) after the completion of cultivating, bacterium solution sample is extracted, the analysis of microorganism group in stomach is carried out using high-throughput sequencing approach；
(5) microorganism reappears in small intestine: intestinal fluid 0.5ml is added in the anaerobism pipe completed with continuous syringe to culture, is added
0.5M sodium hydroxide, adjusting pH are 6.0-7.5；Then anaerobism pipe is placed in constant-temperature table control shaking table temperature is 37 DEG C, oscillation
Frequency 200r/min；Carry out the culture of enteric microorganism group, incubation time 4-6h；In incubation, start to cultivate 30min,
First time exhaust is carried out, in following incubation, is once vented at interval of 1.5h, until cultivating terminates；Exhaust every time
It is centrifuged after the completion, centrifugal speed 8000-12000r/min, centrifugation does not have liquid in managing；It is carried out after the completion of centrifugation in pipe
The supplement of food nutrition liquid and intestinal fluid, and it is 6.0-7.5 that addition sodium hydroxide, which adjusts pH, after cultivating 4h, every 30min is extracted
Bacterium solution measures its absorbance value at 600nm, is that culture terminates according to measured value, when OD value is 0.8-1；
(6) after the completion of cultivating, bacterium solution sample is extracted, the analysis of microorganism group is carried out using high throughput sequencing technologies；
Wherein: gastric juice the preparation method comprises the following steps: 2g NaCl, 3.2g pepsin, are placed in 100mL volumetric flask, it is dense to be added 36.5%
Then HCl 7mL adds sterile distilled water and is settled to 100mL, prepared gastric juice is placed in 4 DEG C of refrigerators and saves；
Pancreatic juice the preparation method comprises the following steps: 0.9g trypsase, 6g bile salt, 12.5gNaHCO3, are dissolved with sterile distilled water and are used in combination
1000mL volumetric flask constant volume, prepared pancreatic juice are placed in 4 DEG C of refrigerators and save；
Food nutrition liquid the preparation method comprises the following steps: proteose peptone (Nanjing Mao Jie microorganism Science and Technology Ltd.) 15g, pancreas casein
Peptone 10g, soy peptone 3g, yeast extract 5g, powdered beef 2g, digestion serum powder 13.5g, beef liver leaching powder 1.2g, glucose 3g,
Dipotassium hydrogen phosphate 2.5g, sodium chloride 3g, soluble starch 0.3g, L-cysteine 0.3g, sodium thioglycollate 0.15g are added 1L and steam
In distilled water, pH is adjusted to 7.2,25 DEG C of 30 min of sterilizing.
4. it is micro- that the system according to claim 3 for reappearing gastrointestinol microorganism group using conditions in vitro carries out reproduction gastrointestinal tract
The method of biology group, it is characterised in that: cultivate 2-4h in the simulation stomach；4-6h is cultivated in simulation small intestine.
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